The first step in establishment of infectious diarrheal disease is the attachment of the invading pathogenic bacteria to the host tissue. In the case of enterotoxigenic Escherichia coli (ETEC), this is mediated by colonization factor antigens (CFA) which are classified by serological type. In human ETEC strains, CFA/I, CFA/II (which includes the CS1 and CS2 pili and the CS3 flexible fibrils), CFA/III and CFA/IV have been defined. These pili are not expressed at temperatures below about 25o, and we have found that CS1 and CS2 expression is positively regulated at the transcriptional level by the product of the plasmid-located rns gene. Recently, we have found that the major CFA/I antigen is positively regulated by a gene (cfaR) homologous to rns, but that other CFA/I strains have no rns homolog. Our goals for the next five years include the definition and characterization of the CS1 and CS2 operons, determination of the gene(s) directly regulated by Rns, elucidation of the mechanism of temperature regulation of CS1 and CS2, determination of whether rns is required for expression of these and other pilin operons, and elucidation of the mechanism of regulation of CFA/I in strains that have no cfaR gene. We hope that the information gleaned from this work will lead to a better understanding of temperature regulation of gene expression and to a strategy for the development of a vaccine to protect against human ETEC strains, or possibly to development of an adhesin analog therapy.